The level of asparagine synthetase activity in most mammalian cell lines is influenced by the concentration of asparagine in the growth medium. A role for asparaginyl-tRNA in the regulation of asparagine synthetase activity is suggested by comparing asparagine synthetase activity in a temperature-sensitive asparaginyl-tRNA synthetase mutant (lys 65a) at permissive and partially restrictive temperatures. At the elevated temperatures there is a 3-fold increase in the level of asparagine synthetase activity. We wll examine the possible role of specific tRNA Asn isoacceptors in CHO cells and in tumor-derived cell lines with altered regulation of asparagine synthetase activity by determining in vivo charging levels of specific isoacceptors under repressed and derepressed conditions. Attempts will also be made to manipulate asparagine synthetase levels in vivo by introducing purified tRNA isoacceptors into cultured cells. Revertants of lys 65a able to grow at elevated temperatures will be isolated and will be characterized both biochemically and genetically. These revertants may include cells with constitutively derepressed levels of asparagine synthetase, with altered transport mechanisms or with other regulatory abnormalities. These revertants will be useful in identifying the molecular participants in the regulation of asparagine synthetase activity levels. Purification of asparagine synthetase and preparation of a specific antibody will enable us to determine if the increased asparagine synthetase levels in CHO cells growing under partially restrictive conditions are due to changes in enzyme content. Alterations in rates of enzyme synthesis and degradation will also be determined. Combined radio-chemical and immunochemical techniques will also be used to characterize the previously described asparagine-dependent tumor cell lines and asparagine-independent variants derived from them.